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Abstract

Background

Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable
enzymes, including glycoside hydrolases and esterases, which are of special interest
for the development of integrated biorefineries. To investigate the strategies used
by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw
(which differ in their chemical composition and tissue organization), were used in
this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass,
the production of enzymes was evaluated and correlated with the initial composition
of the biomass, as well as with the evolution of any residues during growth.

Results

Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also
use wheat bran and straw. The chemical compositions of both lignocellulosic substrates
were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates
in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester
contents of these substrates were also altered. Bacterial growth on both lignocellulosic
biomasses induced hemicellulolytic enzyme production, and xylanase was the primary
enzyme secreted. Debranching activities were differentially produced, as esterase
activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase
production was significantly higher in bacterial cultures grown on wheat bran.

Conclusion

This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources
for enzyme production. The composition of the biomass upon which the bacteria grew
influenced their growth, and differences in the biomass provided resulted in dissimilar
enzyme production profiles. These results indicate the importance of using different
biomass sources to encourage the production of specific enzymes.

Keywords:

Background

The development of biorefineries represents a key advance in access to the integrated
production of bio-derived products, such as energy (fuels, heat), chemicals and materials
[1]. Various starting materials, including agricultural residues (such as sugarcane bagasse,
corn stover, wheat bran (WB) and wheat straw (WS)) and forest residues, represent
biomass substrates of interest to biorefineries [2,3]. The development of integrated biorefineries requires valorizing the entire plant,
and in this context, the transformation of hemicellulosic components of plant cell
walls offers new opportunities for biorefineries to produce high value molecules,
such as alkyl pentosides [4,5].

Lignocellulosic plant cell walls are an assembly of cellulose, lignin and hemicelluloses.
These polymers are linked together by covalent and non-covalent linkages and form
an organized network. Cellulose, main polysaccharide in the plant cell wall, represents
35% to 50% of the dry matter of cell walls [6]. Hemicelluloses represent 25% to 50% of the dry matter in plant cell walls and are
heteropolysaccharides with compositions that vary according to their plant origins.
Arabino-glucurono-xylans are the most abundant hemicelluloses found in graminaceous
plants. They are formed by linear chains of xylans that comprise β-(1,4)-linked D-xylopyranose
residues. These chains can contain various substitute residues, such as L-arabinofuranose,
glucuronic and 4-O-methyl-glucuronic acids, as well as other acetyl groups. In graminaceous plant cell
walls, arabinose residues can be esterified by phenolic compounds, such as ferulic
or p-coumaric acids. Ferulic acid can form diferulic bridges that link two xylan chains
together or associate hemicelluloses to lignin [7,8]. Lignin is a complex phenolic polymer responsible for the rigidity and impermeability
of the plant cell wall, and it represents 10% to 35% of the dry matter in plant cell
walls [6].

The fractionation of lignocellulosic plant cell walls requires the development of
efficient technologies because the complex structures of plant cell walls hinder the
extraction and fractionation of their structural polysaccharides. Several chemical
and physical treatments are able to solubilize and hydrolyze cellulose and hemicelluloses
[9], but the use of biological transformations (including the use of microorganisms and
enzymes) is more beneficial in terms of their energy savings, specificity of action
and environmental friendliness.

In nature, lignocellulosic biomass degradation is carried out by microorganisms (bacteria,
fungi and protozoa) found in various natural ecosystems such as in the digestive tracts
of animals, in soils and in water. Cooperative action occurs between different microorganisms,
allowing for the complete degradation of plant cell walls [10-12]. These microorganisms are able to efficiently degrade cellulose, hemicelluloses and
lignin with the goal of allowing the production of monomeric molecules for use as
carbon sources for growth or for secondary metabolite production. Because lignocellulosic
biomasses are recalcitrant starting materials, their degradation imposes several challenges
for microorganisms, notably the production of a panel of various enzymes to fractionate
polymers constitutive of lignocellulosic biomass. These enzymes are mainly glycoside
hydrolases (cellulases, hemicellulases) and esterases, as well as lignolytic enzymes
produced by some fungi. In the case of the glycoside hydrolases, cooperative action
between endo-enzymes and exo-acting enzymes is required for the liberation of monosaccharides
[13].

One method for developing enzymatic cocktails for the optimal fractionation of polysaccharides
is to gain a better understanding of the dynamic strategies used by the microorganisms
that are able to fractionate polysaccharides and apply this knowledge to the design
of biotechnological processes. Hemicellulases play an important role in efficient
hydrolysis, as these enzymes can be used to create better access to other enzymes
within the cellulose fibrils that are embedded in plant biomass [14,15]. T. xylanilyticus is an aerobic, gram-positive, thermophilic and hemicellulolytic bacterium with no
cellulase activity [16,17]. Four thermostable enzymes have been obtained and characterized from this bacterium:
two xylanases, one arabinofuranosidase and one feruloyl esterase [18-22]. These purified enzymes are able to efficiently degrade plant cell walls, such as
the cell walls of WB and WS [22-26].

The aim of this work was to investigate strategies used by T. xylanilyticus to fractionate wheat bran and straw, two agricultural by-products that are different
in terms of their chemical compositions and tissue organization. The ability of T. xylanilyticus to grow on both of these substrates was studied and compared to its growth on classical
substrates (glucose and xylans). Hemicellulolytic enzyme production was evaluated
and correlated with initial biomass composition, as well as the composition of the
residues produced during growth.

Results

T. xylanilyticus growth kinetics on various substrates

Growth profiles were determined for three different cultures that included monosaccharide
(glucose), polysaccharides (xylan) and two different types of plant cell walls (WB
and WS) by monitoring turbidity of the culture medium for 25 h. Figure 1 shows the growth profiles of T. xylanilyticus based on the various substrates tested. T. xylanilyticus was able to use glucose, xylan and lignocellulosic biomasses as primary carbon sources
for energy production. Growth was rapid on glucose, with a generation time of 111 ± 2
min. On xylan and WB, T. xylanilyticus had comparable generation times (107 ± 9 min and 104 ± 3 min, respectively). However,
its growth on WB showed a significant increase, as its doubling time was higher compared
to that observed for glucose (p < 0.05). T. xylanilyticus was able to use WS as a substrate but did not use it as efficiently as other substrates.
The doubling time on WS was significantly higher (132 ± 4 min) (p < 0.05) than on
other substrates. Growth kinetics curves on glucose, xylan and WB exhibited similar
profiles (Figure 1a-b-c). Exponential phases were reached quickly, either without or after a very short
lag phase, and the stationary phase was defined as an OD600 nm greater than 3 for glucose and xylan and an OD600 nm greater than 1.3 for WB. During the stationary phase, sporulation occurred rapidly
in xylan and glucose cultures (the turbidity of the medium decreased dramatically),
whereas no or few sporulated cells were observed in the WB culture after 13 h. On
WS, the T. xylanilyticus growth profile was again different, following a kinetic curve with two successive
growths. During the first step, absorbance of the culture at 600 nm increased to 0.23.
This step was followed by a long lag phase (4.5 h) and new acceleration and exponential
phases (Figure 1d). The generation time was calculated during the second exponential phase. The stationary
phase was defined as an OD600 nm of greater than 2.5 on WS. Glucose seemed to be a better substrate for use in obtaining
a high quantity of bacterial biomass, as absorbance of the culture on glucose was
approximately 4.2 at the stationary phase. This result corresponded with a cell concentration
of 1.8 × 109 cells/mL. On lignocellulosic substrates, the number of cells obtained was lower (6.1 × 108 cells/mL and 1.1 × 109 cells/mL for OD600 nm 1.6 and 2.5, respectively, for WB and WS substrates). Protein abundance was correlated
to cell growth (Figure 2). A maximal yield was obtained with glucose as a substrate (1972 ± 172 μg/mL), whereas
the culture grown on WB produced only 739 ± 53 μg/mL of proteins). As T. xylanilyticus growths are not identical in the case of the four substrates tested, direct protein
concentration comparisons are not relevant. We normalized protein concentration values
with cultures absorbencies. Thus, WB yielded better protein production (560 ± 40 μg/DO600 nm). Xylan and WS gave equivalent protein yields (316 ± 32 μg/DO600 nm and 301 ± 23 μg/DO600 nm, respectively). Concerning protein localization, a significant increase of excreted
proteins was observed in case of culture on xylan, WB and WS (data not shown).

Figure 1.Growth curves of T. xylanilyticus cultivated on various substrates. Growth was expressed as the ln of absorbance at 600 nm as a function of incubation
time. (a), (b), (c) and (d) show the kinetic curves obtained with glucose, xylan, wheat bran and wheat straw
cultures, respectively. The results are mean values of triplicates, and standard deviations
are less than 5%.

Effect of lignocellulosic composition on T. xylanilyticus growth

WB and WS substrates contained different amounts of carbohydrates (74% and 78% of
the dry matter, respectively). A high content of arabinoxylan was found in WB (51%
of the dry matter) compared to WS (33% of the dry matter). The ratios of arabinose
and xylose residues varied between the two plant materials from 0.72 to 0.14 in WB
and WS, respectively (data not shown). Because the aim of this study was to evaluate
the impact that the chemical composition of the lignocellulosic biomass had on the
growth of T. xylanilyticus, residual carbohydrates, phenolic and acetyl ester contents were analyzed after 24
h, 48 h and 72 h in successive cultures grown on the same plant cell wall materials.
Cell densities in the supernatant phase were also monitored. As shown in Table 1, the carbohydrate contents of WS and WB decreased to 67% and 72%, respectively, after
24 h of growth. The decrease observed in WB was less than that observed in WS; however,
considering the loss of dry matter during culture growth, which was important for
WB (39% during the first 24 h), the decrease in sugar content represented 54% of the
total loss in WB vs. 20% in WS. In accordance with data on total carbohydrates, xylose
and glucose contents were also altered. Taking into account the loss of dry matter,
xylan loss represented 64% and 15% of the loss for WB and WS, respectively, during
24 h of growth. T. xylanilyticus used approximately 26% and 39% of the glucan fraction during 24 h growth on straw
and bran, respectively. Quantification of phenolic ester linked molecules showed a
dramatic decrease in ferulic, p-coumaric and diferulic acid contents during growth on bran, whereas in the case of
straw none of these modifications was observed. Given the residue yields remaining
after growth, the loss of ferulic, p-coumaric and diferulic acids represented 89%, 86% and 73% of the total loss, respectively,
for WB and only 16%, 15% and 21% of the total loss, respectively, for WS. During growth
on WB, 33% of acetyl ester linkages were cleaved after 24 h vs. only 10% for WS. In
parallel, T. xylanilyticus grew very well during the first 24 h as cell concentrations reached 1.0 × 109 cfu/mL for both substrates. During the second and the third cultures, no significant
changes were observed in the chemical composition of the residues. The loss of total
carbohydrates was 10% for both substrates after 48 h of growth. This result represented
a loss of xylose and glucose in WS and a loss of primarily arabinose in WB. In total,
30% and 5% of the acetyl content was liberated from WS and WB, respectively. Despite
this minimal change in chemical composition, growth remained significant (1.0 × 109 cfu/mL and 7.4 × 108 cfu/mL on WB and WS substrates, respectively). After 72 h in culture, growth decreased
drastically as cell densities did not increase beyond 2.7 × 106 cfu/mL on WB and 1.7 × 108 cfu/mL on WS. No significant chemical modifications occurred in the residues after
72 h of growth.

Enzyme production analysis

Endoxylanase activities were observed in the different protein fractions of T. xylanilyticus when the bacterium was cultivated on xylan, WB and WS. As expected, no xylanase activity
was measured in the cultures grown on glucose. Xylanase activity represented the primary
category of enzymatic activity produced by T. xylanilyticus on xylans, WB and WS. The yields obtained were significantly higher on WB cultures
(31.5 ± 4.3 IU/mg) compared to xylan and WS cultures (24.7 ± 2.3 IU/mg and 8 ± 0.46
IU/mg, respectively; P < 0.01 and P < 0.007, respectively) (Figure 3a). As shown in Table 2, xylanases were always highly produced extracellularly, with 86.5%, 82% and 73% of
the total xylanase activity resulting from extracellular production on xylan, WB and
WS, respectively. An increase in cytosoluble xylanase activity was observed during
growth on lignocellulosic biomass (16% and 21% on WB and WS, respectively, compared
to 7% on xylan).

Figure 3.Hemicellulolytic enzyme production by T. xylanilyticus on various substrates during the stationary phase. (a) endoxylanase activity; (b) xylosidase activity; (c) feruloyl-esterase activity; (d) acetyl esterase activity; and (e) arabinofuranosidase activity. Values are the means of samples performed in triplicate
with standard deviation. Activities are expressed as IU/mg of protein or as mIU/mg
of protein and represent the sum of extracellular, cytoplasmic and membrane-associated
activities. G, glucose; X, xylan; WB, wheat bran; and WS, wheat straw.

Table 2.Repartition of enzymatic activities produced by T. xylanilyticus grown on various substrates

Xylosidase activity corresponded to enzymes implicated in the release of monomeric
xylose from xylo-oligosides produced by endo-enzymes. Xylosidase activity ranged between
13.6 ± 4.5 mIU/mg (for glucose) and 99.7 ± 2.8 mIU/mg (for WB). Compared to glucose,
the complex substrates induced greater production of xylosidase. The maximum yield
was obtained with WB (a 7-fold increase compared to the glucose level), whereas increases
were approximately 1.7- and 5-fold on xylan and WS substrates, respectively (Figure
3b). More than 90% of xylosidase activities were found in the soluble protein fraction
of T. xylanilyticus (Table 2).

Debranching enzymes include enzymatic proteins that are able to remove lateral ramifications
from xylan chains. Feruloyl esterase activities were detected in all cultures of T. xylanilyticus (Figure 3c). Measured activities were approximately 41.0 ± 3.9 mIU/mg (glucose) and 235.3 ± 12.8
mIU/mg (WS). Feruloyl esterase activity was also induced in T. xylanilyticus grown in the presence of complex substrates, as increases of 1.4-, 1.8- and 5.7-fold
were observed on xylan, WB and WS substrates, respectively, compared to glucose (P
<0.01; P <0.01; P <0.001). The activities were primarily membrane-associated or occurred
in the soluble protein fractions. On xylan and WB, the induction effect was mediated
by an increase in membrane-associated activities, while on WS, both soluble and membrane-associated
activity levels rose strongly. This led to higher amounts of feruloyl esterase activity
on WS (Table 2).

Acetyl esterases were detected in the intracellular fractions of T. xylanilyticus proteins (Figure 3d). Intracellular activities represented 56%, 79% and 82% of total activities on glucose,
xylan and WB substrate, respectively. Surprisingly in case of WS, the proportion of
activities in the excreted fractions was comparable to the proportion of activities
in the intracellular fractions (58% and 42%, respectively). Compared to glucose, an
induction of 1.66-fold was observed in intracellular acetyl esterase on xylan and
WB (P<0.01). No significant induction of total acetyl esterase activity was observed
with WS; however, an increase of 2.5-fold was detected in excreted acetyl esterase
(P<0.001). The highest activity levels obtained were 365.7 ± 21.8 mUI/mg and 353.0 ± 41.4
mUI/mg on xylan and WB cultures, respectively.

Arabinofuranosidase activities were detected in all cultures of T. xylanilyticus (Figure 3e). These activities were essentially intracellular, as more than 80% of the activity
was detected in the soluble protein fraction of T. xylanilyticus (Table 2). Arabinofuranosidase activity was approximately 40.0 ± 3.0 mIU/mg in cultures grown
on glucose. An induction effect was observed when T. xylanilyticus was grown on complex substrates, with 7- and 1.6-fold increases observed for xylan
and WS, respectively (P < 0.001 and P < 0.01). The maximum induction effect was obtained
with WB, with arabinofuranosidase activity quantified at approximately 420.1 ± 70.0
mIU/mg. This result represented an increase of 10-fold compared to activity measured
in the glucose culture and more than 1.5-fold compared to activity measured in the
xylan culture (P < 0.001 and P < 0.01).

Enzymatic strategies to fractionate bran and straw

To further examine the activity panels produced by the various substrates and to investigate
the mechanism by which T. xylanilyticus fractionates a complex biomass, percentages of each activity obtained on xylan, WB
and WS were compared. If 100% of production represented the sum of all activities,
endoxylanase activity represented the primary hemicellulolytic activity produced by
the bacterial cells. Endoxylanase activity represented 97% of the total enzymatic
activity in cultures grown on xylan and WB. For WS cultures, the amount of xylanase
dropped significantly, to 93.6% of the total activity. This decrease was inversely
correlated with the increase in exo-enzyme activity, with a maximal yield obtained
in the WS culture (6.4% of total production). A focus on accessory enzyme production
showed that on WB, arabinofuranosidase levels increased compared to the WS and xylan
cultures (1.3% vs. 1.1% and 0.8%, respectively), whereas on WS, feruloyl and acetyl
esterase activities represented the largest proportion (5.4% of the total activities)
of exo-enzyme activities. Xylosidase activity was determined to be similar under all
three substrate cultivation conditions.

Discussion

Growth on lignocellulosic substrates and modulation by residue composition

To improve our understanding of feedstock biomass deconstruction by the hemicellulolytic
bacterium T. xylanilyticus, its ability to use destarched WB and WS was studied and compared to its growth on
simple substrates (xylan and glucose). Multiple biological assays were performed with
the aim of providing insight into the physiological behaviors of the bacteria on various
substrates. Our results confirm previous studies that indicated that T. xylanilyticus uses glucose and xylan as its primary carbon sources with equal efficiency [16,17,19]. Similar trends were obtained for the hemicellulolytic bacterium Paenibacillus JDR-2, which utilizes methyl-glucurono-arabinoxylans as well as glucose or xylose,
[27]. In the case of cellulolytic anaerobes such as Clostridium phytofermentans and Caldicellulosiruptor saccharolyticus, growth on hemicellulosic substrates is more efficient than growth on monomeric substrates
[28,29]. We showed that T. xylanilyticus is able to use WB and WS biomass efficiently, as cell densities on this biomass were
greater than 108 cells/mL during the stationary phase. Both cultures exhibited high growth rates and
few sporulation. Yang, et al. showed that Anaerocellum thermophilum was able to use plant cell walls and its growth was similar on switchgrass and poplar
[30]. C. saccharolyticus utilized complex carbohydrates contained in acid-pretreated switchgrass and poplar,
however, the doubling time was significantly higher on the lignocellulosic biomass
compared to xylose or glucose. Indeed, the doubling time on poplar was twofold higher
than the doubling time on pretreated switchgrass, suggesting better utilization of
the latter [29]. Caldicellulosiruptor obsidiansis was also able to use acid-diluted pretreated switchgrass, but its growth rate was
significantly lower compared to growth on cellobiose [31]. Concerning sporulation which occurred later in case of cultures in presence of lignocellulosic
biomass, it is possible that the higher cellular biomass produced on glucose and xylan
led to a rapid use of substrates and then to sporulation. On complex substrates, several
authors observed an accumulation of reducing sugars during stationary phase growth
indicating that what is produced during extracellular degradation may not be assimilated
directly [27,32]. One could suppose that during culture of T. xylanilyticus on lignocelluloses, soluble sugars could be potentially available during a long time
and support a slower growth at the stationnary phase and then delay the sporulation
step. In our study, growth profiles obtained on WB and WS were different as WB was
used more efficiently than WS by T. xylanilyticus. On WS, the doubling time was significantly higher compared with WB, and the growth
kinetics indicated the presence of a double growth. It is possible that an adaptation
phase was needed on WS as the inoculum was produced with xylan cultures. During this
phase T. xylanilyticus cells used soluble compounds in the medium (yeast extract or soluble sugars) to grow.
When those substrates are exhausted, the second lag phase occurred. Xylans in WS are
not very accessible; bacteria could induce the production of hydrolytic enzymes which
are necessary for the fractionation of recalcitrant substrate allowing then the second
growth. This latter one was more efficient. The difference of accessibility and then
difference of growth on straw and bran could be explained by the different compositions
of both lignocellulosic substrates. The low lignin content in WB (3.4%) renders its
xylans and glucans more accessible to T. xylanilyticus[33]. A longer adaptation phase was needed in case of WS which contains high levels of
lignin (20% of the total dry matter) [34]. Analysis of residue compositions after the cultures were grown indicated that the
bacteria were able to use approximately 50% of the total carbohydrates present in
WB after 24 h, whereas the bacteria used only 20% of the total carbohydrates in WS.
An increase in the cultivation time (to 48 h and 72 h) did not improve the utilization
rates of carbohydrates and growth stopped after 48 h in the cultures. This result
suggests that the quantity of non accessible residual carbohydrates to T. xylanilyticus is more important in WS than in WB. The responsiveness to hydrolysis and conversion
of WS could probably be improved by low severity pretreatments such as hot water or
ammonia.

Global protein and enzyme production

Considering the complexity of lignocellulosic feedstocks, the fractionation of these
complex environments represents a difficult challenge for microorganisms. However,
few studies have focused on the correlation between growth, microbial physiology and
enzyme production. In our study, we hypothesized that with plant cell walls as the
primary carbon source, bacterial growth could be correlated with enzyme production
because the enzymes produced are needed to liberate monomeric sugars that support
biomass production. During this study, we observed that T. xylanilyticus was able to produce large quantities of proteins when cultivated on glucose. This
substrate allows for the production of large amounts of biomass, even if the associated
enzymatic activities are low. On a more complex substrate (xylan or lignocellulose),
the amounts of biomass obtained were lower; however, protein production was higher,
particularly the production of excreted and enzymatic proteins. This effect could
be because the majority of bacterial hemicellulolytic systems are inducible and are
controlled by catabolic repression and carbon availability. Such systems enable the
microorganism to adapt rapidly to difficult environments or to complex substrates,
which prevents competition with other organisms [35-37]. T. xylanilyticus is able to adapt its protein production in response to the presence of xylan or more
complex substrates. This adaptation results in the production of a large quantity
of hemicellulolytic enzymes. Cultivation on WB allows for better global enzyme production,
in particular a high level of endoxylanase production. Global enzymatic activities
are lower on WS, and this finding is correlated with lower endoxylanase production.
One explanation could be that in WS cultures, optimal xylanase production does not
occur in the stationary phase. Xylanase, debranching enzyme and xylosidase activities
were detected when T. xylanilyticus was grown on complex substrates, suggesting cooperative action between endo-enzymes
and exo-enzymes. However, xylanase activity was primarily found in the excreted partition
or/and cell associated, whereas debranching enzyme and xylosidase activities were
cell associated or were found in the intracellular fraction. This repartition was
observed earlier in other hemicellulolytic and cellulolytic bacteria. The secretion
of a primary endo-enzyme in the culture medium allowed the oligomerization of polysaccharides
into substituted oligosaccharides which can enter in the cells and are further processed
by membrane or intracellular exo-acting and debranching enzymes. These mechanisms
suggest an ecological strategy employed by these bacteria that prevents the end products
from becoming available to other bacteria. The proximity of resulting hydrolysis products
would decrease diffusion dependant assimilation rates [28,38].

Enzyme production represents a limiting step for lignocellulosic fractionation. Various
studies have shown that using the cellulolytic and hemicellulolytic secretomes of
fungi improves enzyme cocktails [39,40]. The use of bacterial and thermophilic enzymes may present many advantages, as proteins
are often thermostable and resistant to large ranges of pH [41]. Moreover, the use of fungal secretomes could under-represent enzymatic activities,
as lignocellulolytic activities can be intracellular, cell-associated or extra-cellular.
Due to different experimental conditions, it is difficult to compare our results with
those of previous studies. However, it is interesting to note that under our experimental
conditions, the endoxylanase and auxiliary enzyme production obtained from T. xylanilyticus was comparable to or greater than that obtained with the various fungal crude commercial
enzymes evaluated by Chundawat et al. [39].

Strategies used by T. xylanilyticus to fractionate plant cell walls

To provide insight into the biomass degradation strategy used by T. xylanilyticus, our second hypothesis was that on various complex substrates, microorganisms could
produce adaptable enzymes that can more efficiently fractionate their growth substrates
and that changes in enzymatic activity can explain how bacteria attack plant cell
materials. In the literature, most studies have focused on using proteomic analyses
to identify global changes in protein profiles to assess growth on various substrates
or on evaluating endoxylanase production, and studies focusing on the correlations
between enzymatic activities have been rare [31,42]. To verify our hypotheses, we chose to estimate endoxylanase, debranching enzyme
and xylosidase activities during the stationary growth phase of T. xylanilyticus. Endoxylanases are the primary enzymes implicated in hemicellulose conversion by
T. xylanilyticus. However, auxiliary enzyme production profiles differed depending on the growth substrate.
WB and WS did not induce the same enzymatic activities. On WB, the arabinofuranosidase
activity was higher, it is probably necessary to remove the arabinose residues present
in large quantities in this substrate (Ara/Xyl = 0.72). Because, WB cannot have access
to the inner part of the bacterial cell in which arabinofuranosidase activities were
produced, it is possible that induction was due to the presence of soluble oligosaccharides
(substituted or not with arabinose) which are produced by xylanases and could be internalized
into the bacterial cells. Higher production of arabinofuranosidase was accompanied
by a large decrease in arabinose content in bran during the first 24 h of growth.
Arabinose liberation could support bacterial growth after 24 h, when xylose and glucose
are not available. Induction of arabinofuranosidase activity was also observed in
Thermobifida fusca grown on cellulose and lignin [42], indicating that these activities seem to be important for the hydrolysis of lignocellulosic
plant cell walls in which these polymers are embedded with arabinoxylans. Arabinofuranosidase
activity could allow xylanases better access to the xylan components of WB.

Interestingly, when T. xylanilyticus grew on WS, the proportion of esterase activity was higher compared to glucose and
other substrates. Both cytoplasmic and extracellular feruloyl esterase activities
but also extracellular acetyl xylan esterase activities were induced. This is associated
with a high content of acetate and phenolic acids (especially p-coumaric acid) into WS substrate which could induce more feruloyl-esterase enzyme
production. On WB and xylan, feruloyl esterase activity associated with membranes
were induced but in a lesser extent, whereas intracellular acetyl xylan esterase activity
increased noticeably. One can conclude that the esterases implicated in WB fractionation
are not the same as those implicated in WS degradation. Recent studies have shown
that three feruloyl esterases are produced by the hemicellulolytic rumen bacteria
Cellulosilyticum ruminicola H1 [43]. These enzymes differ in substrate specificity and in cellular localization and had
different working patterns when they were associated with xylanases and cellulases.
The authors suggested that they could play a distinct role in lignocellulose degradation
in the rumen. Similar results for acetyl xylan esterase activities were observed in
fungi [44]. Adav et al. showed for the first time that acetyl xylan esterase is upregulated
in the presence of lignin [42]. It is possible that the high content of lignin in straw residues could induce high
levels of esterase production by T. xylanilyticus. The esterase activities produced are implicated either in the cleavage of the feruloyl
groups that link xylans to lignins or link two xylans chains together via diferulic
bridges or in the cleavage of ester bonds between acetyl residues and xylose residues
during the first 24 h of growth.

On WB, high levels of hemicellulolytic enzymes were obtained. The hemicellulolytic
enzyme panels were characterized by the presence of significantly higher arabinofuranosidase
levels. Lower growth and enzymatic production in bacterial cultures grown on WS could
be due to the high quantity of lignin, which could hinder the hemicellulose fraction
and impede the action of the main hemicellulolytic enzymes. However, this limitation
was overcome by the bacterial production of high levels of accessory enzymes such
as esterases.

As WB and WS are too large to pass through the bacterial membrane, small oligosaccharides
released from depolymerization of WS and WB by extracellular or membrane associated
xylanases could act as inducers of cytoplasmic, membrane associated accessory enzymes
or the primary xylanase production observed in T. xylanilyticus cultures. Several studies showed the implication of xylobiose, xylotriose and methylglucuronoxylotriose
in the induction of hemicellulolytic enzymes [35,38]. Since WB and WS had very dissimilar compositions, enzymes profile production and
enzymatic localization, it is possible that inducers are not the same in both cases.
The details of this induction mechanism remain to be elucidated.

Conclusion

An important bottleneck in lignocellulose fractionation is the ability to obtain functional
biocatalysts and to qualitatively and quantitatively define the enzymes necessary
for an optimal balance between biomass deconstruction and enzyme costs. Here, we showed
that T. xylanilyticus actively grows on plant cell walls and produces hemicellulolytic enzymes. These enzymes
are robust, thermostable and able to efficiently fractionate plant cell walls [22,25]. It is clear that the bacterium can adapt its enzymatic profile to better address
the composition of various lignocellulosic substrates. T. xylanilyticus employed concerted enzymatic strategies to grow and to degrade the hemicellulolytic
portion of plant cell walls. Part of its strategy is the utilization of debranching
enzymes. These results are interesting because they provide evidence for the development
of functional enzymatic cocktails that allow for better fractionation of the hemicellulose
portion of plant cell walls and better access to the cellulosic portions of the plant.
We conclude that T. xylanilyticus presents interesting advantages that make it a good model for studying physiological
approaches to enzyme production and lignocellulose degradation, with the aim of developing
the key enzymes.

Methods

Strain and media

Thermobacillus xylanilyticus strain XE, isolated from a manure heap, was used in this study. The strain was available
at the Collection Nationale de Cultures de Microorganismes (France) under the number
CNCM I-1017. The bacterial strain was cultivated on basal medium supplemented with
10% CO2, as described by [17]. For growth kinetics, glucose (Sigma Aldrich, France) solution and destarched wheat
bran and wheat straw (Apache variety, 2 mm) provided by ARD (Pomacle, France) were
autoclaved separately and then added to autoclaved basal medium at a concentration
of 5 g L-1 in 1 L bottles. Oat spelt xylan (Sigma Aldrich, France) was autoclaved together with
the basal medium at a concentration of 5 g L-1.

Growth kinetics on glucose, xylans, wheat straw and bran

Cultures were inoculated with 1% (v/v) of non-sporulated preculture (OD 600 nm = 2 on oat spelt xylan medium) and incubated at 50°C and 130 rpm with glucose, xylan,
bran and straw (as primary carbon sources) in 1 L bottles. Growth was tracked by monitoring
light scattering at 600 nm with a Uvikon 933 spectrophotometer over a period of 24
h. Absorbencies were read directly or after briefly harvesting wheat bran and straw
particles by centrifugation for 30 sec at 500 × g. Samples of culture media containing
the various carbon sources were incubated under the same conditions without inoculation,
and these samples were used as blanks for reading absorbencies. Lag, acceleration,
exponential and stationary phases were determined on a graph representing Ln(OD 600 nm) = f(t). Doubling times (d) were calculated during the exponential phase according
to the formula: n = (Ln(ODt2) – Ln(ODt1))/Ln(2) and d = t2-t1/n where n represents the number of generations. Kinetics experiments were performed
in triplicate, and the means of the doubling time and the standard deviation were
calculated.

Cell counts were made on a Neubauer cell counter using a phase contrast microscope
(Nikon 1528, Japan) with 1000 × magnification, and by inoculating basal-glucose medium
solidified in 15 g agar L-1 with 100 μL of diluted liquid culture (10-6 to 10-8). Cellular concentration was expressed as cells per mL.

Successive growth on straw and bran and residue composition

To evaluate the limitations of cell growth on bran and straw, successive cultivations
with these substrates were performed as described in Figure 4. After the first culture (24 h) on WB and WS, cells were removed and bran and straw
particles were washed three times with sterilized distilled water before the addition
of new basal medium. The reconstituted media were inoculated de novo with 1% (v/v) of non-sporulated precultures as described above. Three successive
cultures were performed on the same WB and WS materials over a period of 72 h. To
evaluate growth, the OD600 nm was measured every 24 h. A negative control (0 h) was prepared by incubating straw
and bran with basal medium at 50°C for 24 h without inoculation. Conversion of WS
and WB was calculated by comparing the initial sugar contents of the untreated bran
and straw with the amount of sugar remaining after 24 h, 48 h and 72 h in presence
of T. xylanilyticus. For this experiment, sugar content of insoluble substrate was evaluated by HPAEC,
after acid hydrolysis as described by [25]. Phenolic acid ester content was evaluated, after alkaline hydrolysis of residues
as described by [22]. The acetyl ester content in the alkaline hydrolysis residues was evaluated using
an acetic acid kit following the manufacturer’s recommendation (Megazyme, Ireland).

Crude protein preparation

After growth on the various substrates described, planctonic cells collected during
stationary phase in 2 mL vials, were separated to the plant particles by centrifugation
for 30 sec at 500 × g, and the three protein fractions were prepared for each condition.
Excreted protein fractions were obtained by centrifugating the complete cultured media
without plant residues at 10,000 × g for 5 min. The supernatant corresponded to the
excreted protein fraction. The pellets of planctonic cells were then resuspended in
the same volume of sodium phosphate buffer (NaH2PO4, pH 7.5), frozen, then sonicated on ice (Vibra-Cell, Bioblock) and centrifuged for
5 min at 10,000 × g. The supernatants contained the intracellular protein fractions.
Cell-associated protein fractions were prepared by solubilizing the residual pellets
in the same volume of phosphate buffer, pH 7.5, supplemented with 0.2% Triton X-100.
Samples were incubated at 4°C overnight and then sonicated. The supernatants, containing
the membrane-associated proteins, were separated from the residual pellets by centrifugation
for 20 min at 20,000 × g. Protein concentrations were determined by the Bradford method
using BSA as a standard [45]. As some soluble proteins could be liberated in the medium from plant residues, protein
concentration in the basal medium incubated with the plant residues alone were substracted
to the protein concentration of extracellular protein.

Endo-xylanase activity

Endo-xylanase activity was assayed in triplicate according to a procedure described
by [46]. An aliquot of 0.1 mL of the extracted proteins was incubated in 0.9 mL birchwood
xylan (Sigma) at 0.5% w/v homogeneously suspended in 50 mM sodium phosphate buffer
(pH 7.5). The xylanase test was performed at 50°C for 10 min. The reducing sugars
were measured by monitoring absorbance at 420 nm every 2 min and by comparison standard
curves describing varying concentrations of xylose. One unit (IU) of enzyme activity
was defined as the quantity of enzyme required to liberate one μmol of equivalent
xylose per minute at 50°C.

Arabinofuranosidase and xylosidase activities

Arabinofuranosidase and xylosidase activities were measured in triplicate by determining
the rate of hydrolysis of p-nitrophenyl α-L-arabinofuranoside (0.5 mM) and p-nitrophenyl β-xylopyranoside (0.5 mM) to p-nitrophenol. Experiments were performed and measured directly using the absorbance
at 401 nm for 5 min at 50 °C with a recording spectrophotometer (Uvikon 933). Reactions
were analyzed in buffered conditions (50 mM sodium phosphate buffer, pH 7.5) with
a total volume of 1 mL containing 0.1 mL of proteins. The extinction coefficient of
pNP (εPNP) was 15,850 M-1.cm-1. All substrates were purchased from Sigma Aldrich (France).

Esterase activities

Feruloyl and acetyl esterase activities were assayed in triplicate against methyl
ferulate (Apin Chemicals, UK) and pNP-acetate (Sigma Aldrich, France), respectively, as described by [22]. The reaction mixture contained 0.1 mL of fractions containing proteins, 0.1 mL of
substrates at 2 mM and 0.8 mL of 50 mM sodium phosphate buffer at a pH of 7.5. The
increase in absorbance at 401 nm (corresponding to acetyl esterase) and the decrease
in absorbance at 340 nm (corresponding to feruloyl esterase) were measured with a
recording spectrophotometer (Uvikon 933) at 50°C for 5 min. The extinction coefficients
of ferulic acid and methyl ferulate are 2,532 M-1.cm-1 and 11,538 M-1.cm-1, respectively, under the assay conditions.

Statistics

The results were compared by statistical analysis with Student’s test. Differences
were deemed significant at a value of p ≤ 0.05.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HR carried out successive growth on various substrates and residues composition analysis;
conceived and organized the study and drafted the manuscript. BH carried out the enzymes
activities measurement and residues composition analysis, NM carried out growth and
enzymes production studies. CR helped to analyze results and to draft the manuscript.
All authors read and approved the submitted version of manuscript.

Acknowledgments

This work was supported by a grant from the University of Reims Champagne Ardenne
(BQR synergies nouvelles 2011). We thank N. Aubry and D. Cronier for analyzing sugar
and phenolic esters composition of wheat bran and wheat straw by HPLC.